2. Definition
Gel electrophoresis is a laboratory method used to separate mixtures of
DNA, RNA, or proteins pushed by an electrical field through a gel that
contains small pores
3. Principle
Gel electrophoresis separates DNA fragments by size in a solid support
medium (an agarose gel). ... The rate of migration is proportional to size:
smaller fragments move more quickly, and wind up at the bottom of the gel.
DNA is visualized by including in the gel an intercalating dye, ethidium
bromide
4. Why do Electrophoresis?
• When DNA is cut by restriction enzymes, the result is a mix of pieces of
DNA of different lengths
• It is useful to be able to separate the pieces - I.e. for recovering particular
pieces of DNA, for forensic work or for sequencing
5. Types of Electrophoresis
• Starch - hydrolyzed potato starch
• Non toxic medium for protein
• Opaque as compare to polyacrylamide
• Agarose gel for DNA separation Run horizontally configuration No
uniform pore size
• Polyacrylamide gel use for protein separation Run vertical configuration
Uniform pore size
6. Material required for Gel Electrophoresis
• Electrophoresis chamber
• Agarose gel
• Gel casting tray
• Buffer
• Staining agent
• Comb
• DNA Ladder
• Sample to be separated
10. • DNA is negatively charge
• When placed in an electric field, DNA will migrate towards positive
pole(anode)
• An Agarose Gel is used to slow the movement of DNA and separate by size
11. How fast will the DNA migrate?
• Small move faster
• Large DNA move slower
• Gel electrophoresis separate DNA according to size
12. Place gel in the electrophoresis chamber
• Add enough electrophoresis buffer to cover the gel
• Make sure each well is filled with buffer
13. Loading the gel
• Carefully place the pippete tip over a well and gently expel the sample
• The sample should sink into well
• Be careful not to puncture the gel with the pippete
14. Running the Gel
• Place cover on the gel electrophoresis chamber
• Connecting the leads to the power supply
• Be sure leads are connected are properly
• DNA migrates towards the anode
• When the power is turned on bubbles should form on the electrodes in the
electrophoresis chamber
15.
16. • After the current is applied make sure gel is running in correct
direction
• Bromophenol blue will run in same direction as DNA
17. Staining Gel
• Ethidium bromide binds to DNA and fluorescent under UV allowing
visualization of DNA on a Gel
• Ethidium bromide can be added to the gel and or running buffer before the
gel can be stained after it has run
• Caution!!!
• Ethidium bromide is a powerful mutagen and is moderately toxic.gloves
should be worn at all time
18. • Place the gel in the staining tray containing warm diluted stain
• Allow the gel to stain for 20-30 minutes
• To remove excess stain allow the gel to destain in water
• Replace water several times for efficient destain
19.
20. Applications
• Estimation of the size of the DNA molecule
• Analysis of the products of the polymerase chain reactions
• Separation of the DNA fragments or extraction and purification
• In the field of the forensic science and molecular biology
